CN114924011A - Method for detecting CMPF content in blood plasma by high performance liquid chromatography tandem mass spectrometry - Google Patents
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Abstract
The invention provides a method for detecting CMPF content in plasma by high performance liquid chromatography tandem mass spectrometry, which comprises plasma sample pretreatment and detection of CMPF content in the pretreated plasma sample by high performance liquid chromatography tandem mass spectrometry. The method has the advantages that the protein precipitation is more complete through the combination of the protein precipitation plate and the organic solvent in the sample pretreatment process, impurities can be effectively removed, the matrix effect is reduced, the precipitation efficiency is improved, the sample pretreatment and detection time are greatly shortened, meanwhile, the organic solvent containing the CMPF isotope internal standard is added in the sample pretreatment, and the interference caused by matrix difference among sample individuals can be effectively overcome.
Description
Technical Field
The invention relates to the technical field of CMPF (China blood plasma factor) detection in plasma, in particular to a method for detecting the CMPF content in plasma by using a high performance liquid chromatography tandem mass spectrometry method.
Background
CMPF is also called 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (3-carboxy-4-methyl-5-propyl-2-furanpropionic acid) is an endogenous metabolite produced by the metabolism of furan fatty acid, and is a major uremic toxin. Recent related research finds that the serum CMPF level in populations with Gestational Diabetes Mellitus (GDM), type 2 diabetes mellitus (T2DM), pre-diabetes mellitus and type 2 diabetes mellitus First Degree Relatives (FDRs) is remarkably increased, and the increase of the content concentration of CMPF can induce glucose intolerance (glucose intolerance), damage glucose-stimulated insulin secretion and reduce the utilization of glucose, so that the insulin secretion of beta cells (also called islet B cells) in pancreas is reduced, and the diabetes mellitus is further developed, and therefore the CMPF can be used as a biochemical index for diabetes mellitus monitoring. Research shows that CMPF has close correlation with IGT, GDM, T2DM and FDRs, but the correlation of CMPF carbohydrate metabolism is not completely clear, so that a method for accurately detecting CMPF is clinically needed, the exact action of CMPF on islet B cells and the specific regulation mechanism thereof are further researched, a new direction can be provided for the treatment of diabetes, and the method has profound significance and wide clinical application prospect.
At present, CMPF detection methods mainly include homogeneous enzyme immunoassay, chemical immunoassay, and high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). However, the inventor of the present application finds, through clinical research, that the homogeneous enzyme immunoassay and the chemical immunoassay have the following defects: firstly, the sensitivity is low, and when the concentration of a sample is very low, a large sample amount is required for enrichment and purification; secondly, the applicability is poor, and the antibody of the object to be detected is easy to generate cross reaction with endogenous substances or metabolites with similar structures, so that a false positive result is caused; thirdly, the analysis efficiency is low, and the sample processing steps are complex. The high performance liquid chromatography-tandem mass spectrometry can avoid the defects of the homogeneous enzyme immunoassay and the chemical immunoassay, but when the UPLC-MS/MS is adopted to detect the sample of the CMPF at present, on one hand, the sample pretreatment time and the detection time are longer, and the longer pretreatment time can limit the clinical application; on the other hand, the sample does not use an isotope internal standard, and the interference caused by matrix difference among sample individuals cannot be effectively overcome.
Disclosure of Invention
The invention provides a method for detecting CMPF content in plasma by high performance liquid chromatography tandem mass spectrometry, aiming at the problems that when the existing UPLC-MS/MS detects a CMPF sample, on one hand, the sample pretreatment time and the detection time are long, so that the clinical application is limited, and on the other hand, the sample does not use an isotope internal standard, so that the technical problem of interference caused by matrix difference among sample individuals cannot be effectively overcome.
In order to solve the technical problem, the invention adopts the following technical scheme:
the method for detecting the CMPF content in the plasma by the high performance liquid chromatography-tandem mass spectrometry comprises the following steps:
plasma sample pretreatment: accurately transferring 100 mu L of plasma sample, placing the plasma sample on a 96-hole protein precipitation plate, adding an organic solvent containing a CMPF isotope internal standard, placing the plasma sample in a constant-temperature oscillator, oscillating the plasma sample for 3min at 700rpm, placing the plasma sample on a positive pressure device, adjusting the flow to be below 40psi, and taking a filtrate as a liquid chromatography tandem mass spectrometry detection sample for later use;
detecting the CMPF content in the pretreated plasma sample by adopting a high performance liquid chromatography tandem mass spectrometry method: the method comprises the steps of firstly separating CMPF by using high performance liquid chromatography, then quantifying by using a mass spectrum isotope internal standard method, establishing a correction curve by using the concentration ratio of a CMPF target compound and a CMPF isotope internal standard as an X axis and the peak area ratio of the CMPF target compound and the CMPF isotope internal standard as a Y axis, and calculating the content of CMPF in plasma, wherein the specific instrument conditions are as follows:
(1) high performance liquid chromatography conditions:
mobile phase A: 5mM ammonium formate water;
mobile phase B: 0.1% formic acid + methanol;
type of chromatographic column: waters UPLC HSS C18SB 2.1.1 mm X50 mm,1.8 μm
The column temperature is 40 ℃ plus or minus 5 ℃, the sample injection amount is 10 mu L, the temperature of a sample injection disc is 10 ℃ plus or minus 5 ℃, and the flow rate is 0.4 mL/min;
a gradient elution mode was used, as shown in table 1 below:
TABLE 1 gradient elution procedure
(2) Mass spectrum conditions:
ionization mode: in an electrospray positive ion mode, a mass spectrum scanning mode of multi-reaction monitoring is adopted, and the voltage of a capillary is 4.0 kV; the ion source temperature is 150 ℃; the temperature of the desolvation gas is 650 ℃; the flow rate of the desolventizing gas is 650L/Hr; simultaneously monitoring the target compound and the isotope internal standard;
multiple reaction monitoring mode, ion parameters: CMPF: 239.1596 is more than 151.114, the taper hole voltage is 32V, and the collision energy is 16V; CMPF-d 3: 242.1596>154.173, the cone voltage is 32V and the collision energy is 18V.
Further, an organic solvent containing a CMPF isotope internal standard is added in the plasma sample pretreatment, and the preparation method is as follows: preparing 1mg/mL CMPF-d3 standard stock solution by using methanol as a solvent, and preparing an organic solvent containing 1.0 mu g/mL CMPF-d3 by using the methanol.
Further, the CMPF target compound is formulated as follows: preparing a CMPF standard stock solution with the concentration of 1mg/mL by using methanol as a solvent, preparing a mixed standard solution with CMPF concentrations of 0.02 mu g/mL, 0.1 mu g/mL, 0.5 mu g/mL, 1.0 mu g/mL, 5.0 mu g/mL, 10.0 mu g/mL and 50 mu g/mL respectively by using a 5% m/v BSA solution, subpackaging the mixed standard solution in 7 gradients of 1.5mL brown bottles, and storing at-20 ℃ for later use.
Compared with the prior art, the method for detecting the CMPF content in the blood plasma by the high performance liquid chromatography-tandem mass spectrometry provided by the invention has the following advantages:
1. the invention establishes a method for quickly detecting the content of CMPF in a plasma sample at high flux by optimizing the conditions of sample pretreatment and high performance liquid chromatography tandem mass spectrometry, and is a detection method with simple sample pretreatment, high flux and reliable result.
2. In the sample pretreatment process provided by the invention, through the combination of the protein precipitation plate and the organic solvent, compared with the method of simply using the organic solvent as a precipitator, the protein precipitation is more complete, impurities can be effectively removed, the matrix effect is reduced, the precipitation efficiency is improved, the sample pretreatment and detection time is greatly shortened, the treatment time of 96 samples can be completed in 10min, the analysis of a single sample can be completed within 2min, and the CMPF content can be rapidly and sensitively detected.
3. In the sample pretreatment, an isotope internal standard with chemical properties basically consistent with those of CMPF is added into an organic solvent, so that on one hand, the deuterium position of the isotope internal standard does not interfere with the accurate quantification of CMPF, and on the other hand, the interference caused by matrix difference between sample individuals can be effectively overcome.
Drawings
FIG. 1 is an ion flow diagram of CMPF and its internal standard in a standard provided by the present invention.
FIG. 2 is a CMPF line graph established using isotopic internal standard quantitation methods provided by the present invention.
Figure 3 is an ion flow graph of CMPF and its isotopic standard in an actual plasma sample provided by the present invention.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further explained below by combining the specific drawings.
The invention provides a method for detecting CMPF content in blood plasma by high performance liquid chromatography tandem mass spectrometry, which comprises the following steps:
(1) CMPF target compound formulation: preparing CMPF standard stock solution with 1mg/mL by using methanol as a solvent, preparing mixed standard solution with CMPF concentrations of 0.02 [ mu ] g/mL, 0.1 [ mu ] g/mL, 0.5 [ mu ] g/mL, 1.0 [ mu ] g/mL, 5.0 [ mu ] g/mL, 10.0 [ mu ] g/mL and 50 [ mu ] g/mL by using 5% m/v BSA (bovine serum albumin) solution, respectively, distributing the mixed standard solution into 1.5mL brown bottles, and storing the brown bottles at-20 ℃ for later use.
(2) Preparing an organic solvent: preparing 1mg/mL CMPF-d3 standard stock solution by using methanol as a solvent, and preparing an organic solvent containing 1.0 mu g/mL CMPF-d3 by using the methanol.
(3) Plasma sample pretreatment: accurately transferring 100 mu L of plasma sample, placing the plasma sample on a 96-pore protein precipitation plate, adding an organic solvent containing a CMPF isotope internal standard, placing the plasma sample in a constant-temperature oscillator, oscillating the plasma sample for 3min at 700rpm, placing the plasma sample on a positive pressure device, adjusting the flow to be below 40psi, and taking the filtrate as a liquid chromatogram tandem mass spectrometry detection sample for later use, namely preparing for liquid chromatogram tandem mass spectrometry detection.
(4) Detecting the CMPF content in the pretreated plasma sample by adopting a high performance liquid chromatography tandem mass spectrometry method: firstly, separating CMPF by using high performance liquid chromatography, then quantifying by using a mass spectrum isotope internal standard method, establishing a correction curve by taking the concentration ratio of a CMPF target compound to the CMPF isotope internal standard as an X axis and the peak area ratio of the CMPF target compound to the CMPF isotope internal standard as a Y axis, and calculating the content of CMPF in plasma, wherein the specific instrument conditions are as follows:
high performance liquid chromatography conditions:
a mobile phase A: 5mM ammonium formate water;
mobile phase B: 0.1% formic acid + methanol;
the type of the chromatographic column: waters UPLC HSS C18SB 2.1.1 mm. times.50 mm,1.8 μm
The column temperature is 40 ℃ plus or minus 5 ℃, the sample injection amount is 10 mu L, the temperature of the sample injection disc is 10 ℃ plus or minus 5 ℃, and the flow rate is 0.4 mL/min;
a gradient elution mode was used, as shown in table 1 below:
TABLE 1 gradient elution procedure
Wherein, Curve in the above Table 1 refers to a Curve type, i.e., the flow phase ratio is increased in a linear manner;
mass spectrum conditions:
ionization mode: in an electrospray positive ion mode, a mass spectrometry scanning mode of Multiple Reaction Monitoring (MRM) is adopted, and the Capillary voltage (Capillary) is 4.0 kV; the ion source temperature is 150 ℃; desolvation temp. is 650 deg.C; the desolventizing gas flow rate (Desolvation) was 650L/Hr; simultaneously monitoring a target compound and an isotope internal standard, and optimizing the taper hole voltage and the collision voltage of each target object respectively so as to achieve higher stability and sensitivity;
multiple Reaction Monitoring (MRM) mode, ion parameters: CMPF: 239.1596>151.114 (quantitative ion pairs), the cone voltage is 32V, and the collision energy is 16V; CMPF-d3 (IS): 242.1596>154.173, the cone voltage is 32V and the collision energy is 18V.
(5) And (3) sample test results:
(6) product performance test data:
first, a total ion flow diagram of CMPF and its isotopic internal standard in standards and samples: the CMPF standard and plasma samples have symmetrical peak patterns, and have substantially no interference from spurious peaks at concentrations no greater than 50 μ g/mL, indicating that good detection can be obtained under these conditions, as shown in fig. 1 and 3.
Second, calibration curve: by adopting an isotope internal standard quantitative method, the concentration ratio of a CMPF target compound (standard substance) to a CMPF isotope internal standard (internal standard substance) is used as an X axis by utilizing the existing MasstLynx software, the peak area ratio of the CMPF target compound (standard substance) to the CMPF isotope internal standard (internal standard substance) is used as a Y axis, a correction curve is established, and the concentration of a substance to be detected in plasma is calculated. The linear fitting equation of CMPF is good in linearity in the corresponding concentration range, the correlation coefficient is above 0.995, and the quantitative requirements are met, which is shown in FIG. 2.
Third, adding standard recovery rate experiment: collecting low-concentration mixed human plasma, taking 10 mu L of standard solution and 90 mu L of mixed plasma, and preparing samples with theoretical concentrations of 100ng/mL, 500ng/mL, 5000ng/mL and 10000ng/mL in parallel by 6 parts; after vortex mixing, the operation is carried out according to the sample pretreatment steps, and the determination results are shown in the following table:
| standard concentration (ng/mL) | Recovery(％) | RSD(％，n＝6) |
| 100 | 81.40 | 2.15 |
| 500 | 104.04 | 2.93 |
| 5000 | 118.81 | 2.15 |
| 10000 | 94.79 | 2.32 |
As can be seen from the table above, the Recovery rate (Recovery) of each spiked concentration is 80-120%, the Relative Standard Deviation (RSD) is within 3%, and the repeatability is good.
Compared with the prior art, the method for detecting the CMPF content in the blood plasma by the high performance liquid chromatography tandem mass spectrometry has the following advantages:
1. the invention establishes a method for rapidly detecting the CMPF content in the plasma sample at high flux by optimizing the sample pretreatment and high performance liquid chromatography tandem mass spectrometry conditions, and is a detection method with simple sample pretreatment, high flux and reliable result.
2. In the sample pretreatment process provided by the invention, through the combination of the protein precipitation plate and the organic solvent, compared with the method of simply using the organic solvent as a precipitator, the protein precipitation is more complete, impurities can be effectively removed, the matrix effect is reduced, the precipitation efficiency is improved, the sample pretreatment and detection time is greatly shortened, the treatment time of 96 samples can be completed in 10min, the analysis of a single sample can be completed within 2min, and the CMPF content can be rapidly and sensitively detected.
3. In the sample pretreatment, the isotope internal standard with chemical property basically consistent with that of CMPF is added in the organic solvent, so that on one hand, the deuterium position of the isotope internal standard does not interfere with the accurate quantification of CMPF, and on the other hand, the interference caused by matrix difference between sample individuals can be effectively overcome.
Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.
Claims (3)
1. The method for detecting the CMPF content in the plasma by the high performance liquid chromatography-tandem mass spectrometry is characterized by comprising the following steps of:
plasma sample pretreatment: accurately transferring 100 mu L of plasma sample, placing the plasma sample on a 96-hole protein precipitation plate, adding an organic solvent containing a CMPF isotope internal standard, placing the plasma sample in a constant-temperature oscillator, oscillating the plasma sample for 3min at 700rpm, placing the plasma sample on a positive pressure device, adjusting the flow to be below 40psi, and taking a filtrate as a liquid chromatography tandem mass spectrometry detection sample for later use;
detecting the CMPF content in the pretreated plasma sample by adopting a high performance liquid chromatography tandem mass spectrometry method: the method comprises the steps of firstly separating CMPF by using high performance liquid chromatography, then quantifying by using a mass spectrum isotope internal standard method, establishing a correction curve by using the concentration ratio of a CMPF target compound and a CMPF isotope internal standard as an X axis and the peak area ratio of the CMPF target compound and the CMPF isotope internal standard as a Y axis, and calculating the content of CMPF in plasma, wherein the specific instrument conditions are as follows:
(1) high performance liquid chromatography conditions:
mobile phase A: 5mM ammonium formate water;
mobile phase B: 0.1% formic acid + methanol;
the type of the chromatographic column: waters UPLC HSS C18SB 2.1.1 mm. times.50 mm,1.8 μm
The column temperature is 40 ℃ plus or minus 5 ℃, the sample injection amount is 10 mu L, the temperature of a sample injection disc is 10 ℃ plus or minus 5 ℃, and the flow rate is 0.4 mL/min;
a gradient elution mode was used, as shown in table 1 below:
TABLE 1 gradient elution procedure
(2) Mass spectrum conditions:
ionization mode: in an electrospray positive ion mode, a mass spectrum scanning mode of multi-reaction monitoring is adopted, and the capillary voltage is 4.0 kV; the ion source temperature is 150 ℃; the temperature of the desolvation gas is 650 ℃; the flow rate of the desolventizing gas is 650L/Hr; simultaneously monitoring the target compound and the isotope internal standard;
multiple reaction monitoring mode, ion parameters: CMPF: 239.1596 is more than 151.114, the taper hole voltage is 32V, and the collision energy is 16V; CMPF-d 3: 242.1596>154.173, the cone voltage is 32V and the collision energy is 18V.
2. The method for detecting the CMPF content in the plasma by the high performance liquid chromatography-tandem mass spectrometry according to claim 1, wherein an organic solvent containing an internal standard of a CMPF isotope is added in the pretreatment of the plasma sample, and the method is as follows: preparing 1mg/mL CMPF-d3 standard stock solution by using methanol as a solvent, and preparing an organic solvent containing 1.0 mu g/mL CMPF-d3 by using the methanol.
3. The method for detecting the CMPF content in the plasma by high performance liquid chromatography-tandem mass spectrometry according to claim 1, wherein the CMPF target compound is prepared by the following method: preparing a CMPF standard stock solution with the concentration of 1mg/mL by using methanol as a solvent, preparing a mixed standard solution with the CMPF concentrations of 0.02 mu g/mL, 0.1 mu g/mL, 0.5 mu g/mL, 1.0 mu g/mL, 5.0 mu g/mL, 10.0 mu g/mL and 50 mu g/mL respectively by using a 5% m/v BSA solution, subpackaging the mixed standard solution in a brown bottle with 1.5mL, and storing the mixed standard solution at-20 ℃ for later use.
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| CN104822378A (en) * | 2012-09-21 | 2015-08-05 | 多伦多大学理事会 | CMPF as a biomarker for diabetes and associated methods |
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| CN114152743A (en) * | 2021-12-02 | 2022-03-08 | 沈阳易康元血液透析科技有限公司 | Chemiluminescence immunoassay kit for detecting CMPF and detection method thereof |
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| CN104822378A (en) * | 2012-09-21 | 2015-08-05 | 多伦多大学理事会 | CMPF as a biomarker for diabetes and associated methods |
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| CN110907569A (en) * | 2019-12-25 | 2020-03-24 | 山东英盛生物技术有限公司 | Method for simultaneously detecting 4 protein-bound uremia toxins in blood sample |
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| 任真真等: "蛋白结合类尿毒症毒素蛋白结合率方法测定", 空军医学杂志, vol. 31, no. 5, pages 284 * |
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